In digital microscopes (in this patent application, “digital microscope” includes, without limitation, microscopes in conjunction with an imaging system), a specimen image that has been magnified by an objective lens is magnified and captured by a built-in digital camera, and then is displayed on monitor device. There tends to be a demand for a microscope user to make size measurements directly on displayed images. To this end the following two basic methods may be used.
One way is called theoretical calibration/scaling, in which a total magnification is calculated by multiplying the magnification of the objective (generally engraved in the objective body) by the magnification of the camera adaptor lens and the magnification of the monitor device. The real size of the specimen is derived by dividing the display image size (measured with software) by the total magnification. The drawbacks of this method are: the user needs to know which components are currently installed at the microscope, and needs to know the influence on total magnification of every exchangeable microscope component—and how they interact with each other. Another disadvantage is that theoretic scaling cannot take into account an individual device's production tolerances.
Another way is measuring calibration/scaling, in which the digital microscope software provides a pixel mapping function to measure a pixel distance of the image. To transfer a pixel distance to a real distance on the specimen, the operator needs to measure an object of known size for comparison. Via the reference object, the relationship between pixel distance on the monitor screen and the real distance on specimen itself can be accurately summed and recorded by the microscope software. After that, the calibrated system can be used to measure the specimen of interest. The reference object mentioned above is called a calibration plate. Measuring scaling does not require much knowledge of the user's microscope on the side of the microscopy software. It does require, however, the user to have a reference object of a known real size, which can then be imaged by the microscopy software. The software then measures the pixel distance between two features in that image, and with the help of the user who enters the real (known) distance, mapping is established.
A typical calibration plate most widely used in the market includes a graduated ruler pattern. When the measuring calibration procedure starts, operator places the calibration plate on the microscope stage. Then he/she chooses an objective, focuses and moves the calibration plate until the ruler image appears clearly in the monitor device. Thereafter the operator selects a ruler start and an end point on the monitor, and the software calculates the pixel distance (D_pixel) therebetween. The operator counts the ruler divisions on the monitor, (i.e. the number of intervals between selected start and end point). Since one division distance on the ruler is known, the real distance (D_real) between the selected start and end point can be calculated. The magnification M can be derived by dividing the pixel distance by the real distance (M=D_pixel/D_real) and saved in the microscope software, and thereby the measuring calibration of the chosen objective is completed. Finally the operator may change to other objectives and repeat the above steps until all the objectives are calibrated. The drawbacks of the above measuring calibration by using the ruler are: the calibration process needs too much user intervention (selecting ruler start and end point), and the calibration accuracy depends on user operation. It will impact calibration accuracy if the user measures reference objective wrongly.
As an example of measuring calibration, it is proposed in a reference document (M. T. Postek, Critical Issues in Scanning Electron Microscopes Metrology, Journal Of Research of the National Inst. of Standards & Technol., Vol. 99, No. 5, Oct. 1994, pp. 658-660) to use pitch magnification as a standard for magnification calibration. This provides significant advantages in the precision of a microscope's magnification calibration, as the pitch reference contains several repeatable identical features (lines or stripes). Independently of the type or model of the microscope being calibrated, these patterned lines will appear to be identical to each other. This strongly facilitates evaluation of the pitch value of structures present in the microscope image—specifically, the distance between any equivalent points of adjacent stripe pattern features in the image can be considered as the pitch value. Such points can be established or noted by using the maxima or minima of brightness in the video signal, and any repeated characteristic features in the video signal—slopes, etc. In such a method, the pitch value is obtained on the basis of the signal intensity—maxima and minima of brightness in the image, and is not a precise algorithm based on geometrical features, because the brightness tends to be affected by various errors.
As another example of measuring calibration, it is described in patent application US2005220362 an improved method of precision calibration of a microscope magnification including calculating a magnification scale as a quotient obtained when an image size of a test object viewed or collected with the microscope as divided by a true test object size. The method comprises the steps of obtaining a magnification reference by taking a diffraction grating with a tested pitch value as the test object; distributing a brightness level between 30-70% amplitude in one of an image of the diffraction grating and a video signal obtained in the microscope; calculating a position of the video signal “center of mass” for each of formed “islands” of the brightness distribution; considering an average distance between neighboring “center of mass” as a grating pitch in a microscope image of the object; and recognizing that a magnification scale of the microscope is a result of a division of an average pitch dimension by the true grating pitch. The drawback in this method is that magnification is only calculated in one dimension, and “center of mass” signal is prone to be impacted by error sources. Thus, the accuracy and robustness of the method is limited.
In JP2004078162, a magnification calculation method is disclosed. The principle of the measurement is to let a round object of known size superimpose with circular reticle in front of the eyepiece to determine magnification. This simple method nevertheless has some notable drawbacks: it is not an automatic measuring calibration, is only suitable for a continuous zoom microscope, and has low accuracy.